PROJECT SUMMARY Transition metal ions are essential nutrients for all organisms. The availability of these nutrients plays a critical role in the host-microbe interaction and microbial pathogenesis. The primary objective of this research proposal is to elucidate how the host-defense protein calprotectin (CP) sequesters transition metals from microbes and thereby contributes to the innate immune response. CP provides a remarkable example of unique biological coordination chemistry that is relevant to infectious disease and microbial pathogenesis. Each CP heterodimer (S100A8/S100A9) exhibits six different sites for chelating divalent cations, including calcium (Ca) and transition metals. Our central hypothesis is that CP responds to physiological Ca(II) gradients to tune its coordination chemistry for transition metals and to modulate its biological function as an antimicrobial protein that deprives invading pathogens of essential nutrient metals (e.g. manganese, iron, zinc). The proposed investigations are based on preliminary data that Ca(II) binding by human CP (hCP) at the EF- hand domains triggers high-affinity chelation of transition metals at sites formed at the interface of the S100A8 and S100A9 subunits. In Aim 1, we will investigate how Ca(II) ions modulate hCP structure and tune its affinities for transition metals. In Aim 2, we will evaluate how the murine orthologue (mCP) sequesters transition metals and thereby provide needed molecular and biophysical insights into literature results of CP from animal models of infection. In Aim 3, we will investigate the competition between CP and bacterial metal- transport machinery for manganese(II). These fundamental bioinorganic and biophysical initiatives constitute an innovative departure from biological and medical studies of CP, and highlight the importance of applying quantitative analytical and spectroscopic methods to a problem central to human health and disease. Taken together, the results will provide new molecular insights into how CP contributes to innate immunity and metal homeostasis. Moreover, the ability to acquire metal ions is an important facet of microbial pathogenesis, and both intercepting microbial metal acquisition and boosting the metal-withholding response of the host present opportunities for antibiotic development. We anticipate that the results from our work will, in the long term, help to guide the development of new antimicrobial therapeutics that target these processes central to the host- pathogen interaction.